Aggregability of the SQSTM1/p62-based aggresome-like induced structures determines the sensitivity to parthanatos

Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) triggers a noncanonical form of programmed cell death (PCD) called parthanatos, yet the mechanisms of its induction are not fully understood. We have recently demonstrated that the aggresome-like induced structures (ALIS) composed of the autophagy receptor SQSTM1/p62 and K48-linked polyubiquitinated proteins (p62-based ALIS) mediate parthanatos. In this study, we identified the D1 dopamine receptor agonist YM435 as a unique parthanatos inhibitor that acts as the disaggregating agent for the p62-based ALIS. We found that YM435 structurally reduces aggregability of the ALIS, and then increases its hydrophilicity and liquidity, which prevents parthanatos. Moreover, dopamine and L-DOPA, a dopamine precursor, also prevented parthanatos by reducing the aggregability of the ALIS. Together, these observations suggest that aggregability of the p62-based ALIS determines the sensitivity to parthanatos, and the pharmacological properties of YM435 that reduces the aggregability may be suitable for therapeutic drugs for parthanatos-related diseases such as neurodegenerative diseases.

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), are diseases in which neuronal function declines due to loss or death of nerve cells, leading to various neurological disorders [14].Since aging is a risk factor of the neurodegenerative diseases, the number of patients accompanying aging population is expected to increase explosively [15].However, no effective therapeutic treatment has been established to prevent the progression of neurological dysfunction, and therefore its development is an urgent issue in modern medicine.As mentioned above, accumulating evidence indicates that parthanatos is involved in the neurologic deficits in the neurodegenerative diseases [5][6][7][8][9].In particular, protein aggregates responsible for triggering neurodegenerative diseases (e.g., aggregation of amyloid β, α-synuclein, and TDP-43) initiate parthanatos through oxidative stress.Therefore, parthanatos has emerged as an attractive therapeutic target for the neurodegenerative diseases.
Our cellular experiments have demonstrated that protein misfolding caused by oxidative stress promotes the formation of the p62-based ALIS composed of the autophagy receptor SQSTM1/p62 and K48-linked polyubiquitinated proteins, which triggers parthanatos [16].Although molecular mechanisms by which the p62-based ALIS are involved in the PARP-1-dependent cell death machinery are still unclear, poly-ADP-ribosylation (PARylation) of the p62-based ALIS mediated by PARP-1 may play a key role in parthanatos (unpublished data).On the other hand, we have recently demonstrated that supersulfides dissolve the p62-based ALIS, and thereby inhibited subsequent parthanatos [17].Mechanistically, the supersulfide donor Na 2 S 4 promotes the persulfidation of heat shock protein 90 (HSP90) at Cys521, leading to the activation of heat shock factor 1 (HSF1).The activated HSF1 transcriptionally upregulated HSP70, which promotes disaggregation of the p62-based ALIS [18].Therefore, the stability of the p62based ALIS seems to determine the sensitivity to parthanatos.In addition, considering that p62 is included in the neurodegenerative disease-related aggregates, such as Levy's body, Huntington's aggregate, and in the neurofibrillary degeneration in Alzheimer's disease, targeting the p62-related aggregates may be effective for the treatment of neurodegenerative diseases [19][20][21][22][23].
It has been reported that various compounds containing the catechol skeleton, such as polyphenols and catecholamines, can suppress the protein aggregate formation in both in vitro and in vivo [24][25][26].Although the detailed mechanisms by which these compounds dissolve the protein aggregates are still unknown, compounds, such as polyphenols, that act as both antioxidants and disaggregating agent may be highly effective for the treatment of the neurodegenerative diseases.YM435, a smallmolecule compound that has catechol skeleton, has been reported as a selective agonist of the D1 dopamine receptor [27].By acting as a D1 dopamine receptor agonist, YM435 promotes vasodilatory effects and exocrine pancreas [27][28][29][30], and has been found to improve renal and heart failure at least in model animals [31][32][33][34].In this study, we newly found that YM435 acts as both the antioxidant and disaggregating agent, and strongly inhibits parthanatos.Thus, our findings may provide insight into the novel therapeutic approaches for neurodegenerative diseases.

YM435 suppresses oxidative stress-induced parthanatos by acting as an antioxidant
We have previously demonstrated that hydrogen peroxide (H 2 O 2 ) preferentially initiates oxidative stress-induced parthanatos in the human fibrosarcoma HT1080 cell line [16].As shown in Fig. 1A, H 2 O 2 causes PARP1-dependent poly-ADPribosylation (PARylation), showing that H 2 O 2 stimulates the PARP-1 activation.Reduced cell viability upon H 2 O 2 treatment was completely restored in PARP-1 knockout (KO) HT1080 cells (Fig. 1B), or by the treatment with the conventional PARP-1 inhibitors (Fig. 1C).Collectively, these data confirm that H 2 O 2 induces parthanatos in HT1080 cells.Interestingly, we newly found that YM435 inhibited the reduction of cell viability induced by H 2 O 2 (Fig. 1D), and therefore examined whether YM435 inhibits parthanatos.As shown in Fig. 1E and F, YM435 inhibited the PARP-1 activation, and the nuclear translocation of AIF triggering large-scale DNA fragmentation, an index of the parthanatos induction, triggered by H 2 O 2 .YM435 also inhibited the accumulation of PARP-1 in the chromatin fraction required for parthanatos induction (Fig. 1G) [35,36].Moreover, comet assay revealed that YM435 inhibits oxidative stressinduced DNA damage, when comet scores were calculated as previously described (Fig. 1H) [37].YM435 also inhibited the ROS accumulation (Figs.1H and 1I).Therefore, these observations suggest that YM435 suppresses oxidative stress-induced parthanatos by acting as an antioxidant.

Antioxidant activity of YM435 is effective in ex vivo
It is known that neuronal damage and death triggered by oxidative stress are major causes of neurodegenerative diseases.Therefore, the antioxidant activity of YM435 may be potentially effective for prevention of neurodegenerative diseases triggered by neuronal damage and death.We therefore investigated the inhibitory effect of YM435 in neuronal cells.As shown in Fig. 2A, YM435 clearly inhibited the reduction of cell viability induced by H 2 O 2 to a similar extent as the PARP-1 inhibitor DPQ or pamiparib treatment in SH-SY5Y cultured neural cells.In addition, similar results were seen in PC12 cells (Figs. 2B and 2C).We next examined whether YM435 can cross the blood-brain barrier (BBB) to evaluate the neuronal protective effect of YM435 in vivo.However, brain concentrations of YM435 were much lower than veliparib that can cross the BBB [9], when 1 mM YM435 or veliparib dissolved in PBS was infused into the jugular vein of C57BL mice for 1 hour (Fig. 2D).We therefore examined the neuroprotective effects of YM435 ex vivo to perform analysis under conditions closer to those of living organisms.In mouse brain slices, propidium iodide (PI) staining revealed that rucaparib, and antioxidants such as propyl gallate and N-acetylcysteine significantly reduced PI-positive (dead) cells induced by H 2 O 2 , suggesting that H 2 O 2 induce oxidative stress-mediated parthanatos in mouse brain slices (Fig. 2E and F).Interestingly, we found that YM435 clearly inhibited H 2 O 2 -induced parthanatos in mouse brain slices (Fig. 2G).Moreover, YM435 successfully prevented oxidative stress-mediated cell death induced by amyloid β-peptide (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) that shows a cytotoxic effect on neuronal cells, which is associated with the pathogenesis of Alzheimer's disease (Fig. 2H) [38,39].Thus, YM435 cannot cross the BBB, but its antioxidant activity is effective in ex vivo.

YM435 suppresses parthanatos independently of its antioxidant activity
We next confirmed that YM435 is effective for parthanatos induced by cefotaxime (CTX), a third-generation cephalosporin antibiotic that promotes reactive oxygen species (ROS) generation [40,41], that particularly triggers parthanatos mediated by the p62-based ALIS [16].As shown in Figs.3A and 3B, CTX-induced parthanatos was clearly inhibited by PARP-1 knockout, and the PARP-1 inhibitors as previously described [17].Interestingly, we noticed that YM435 inhibits parthanatos induced by CTX with much lower concentrations (Fig. 3C), when compared with H 2 O 2 (Fig. 1D).Moreover, the lower concentrations of YM435 (10 and 20 µM) did not affect CTX-induced ROS generation (Fig. 3D).Our previous studies have demonstrated that CTX-induced ROS generation causes the NF-E2-related factor 2 (Nrf2) activation, which upregulates quinone acceptor oxidoreductase 1 (Nqo1), a well-known target of Nrf2, and promotes the accumulation of p62 protein [41].Consistent with the observation that YM435 did not affect CTX-induced ROS generation, the nuclear translocation of Nrf2 induced by CTX was not affected by the YM435 treatment (Fig. 3E).Moreover, the induction of Nqo1 and p62, and the accumulation of p62 protein were not affected by the YM435 treatment (Fig. 3F and G).Collectively, these results suggest that YM435 suppresses CTX-induced parthanatos independently of its antioxidant activity.

YM435 reduces aggregability of the p62-based ALIS
We next examined how YM435 suppresses CTX-induced parthanatos without affecting the ROS generation.We therefore examined whether YM435 affects the formation of the p62based ALIS, since CTX-induced parthanatos is mediated by the p62-based ALIS that is biochemically depicted as the accumulation of p62 and K48-linked polyubiquitinated proteins in the Triton-X-insoluble fraction [16].Therefore, p62 is required for CTXinduced parthanatos as previously described (Fig. 4A) [16].YM435 apparently reduced the accumulation of p62 and K48-linked polyubiquitinated proteins in the Triton-X-insoluble fraction (Fig. 4B), without changing the total amount of the p62 and polyubiquitinated proteins (Figs. 3G and 4C), suggesting the possibility that YM435 suppresses parthanatos by preventing the ALIS formation.On the other hand, the p62-ALIS is microscopically observed as costained puncta of p62 and K48-linked ubiquitin [16].Given that YM435 inhibits the ALIS formation, the fluorescent puncta of p62 and K48-linked ubiquitin are reduced by the YM435 treatment.However, both the number and size of the fluorescent puncta of p62 and K48-linked ubiquitin were not affected by the YM435 treatment (Figs. 4D and 4E).To explain the discrepancy between the biochemical and microscopic analyses, we speculated that YM435 increases the hydrophilicity of the p62-based ALIS rather than inhibition of its formation.Of note, recent evidence has demonstrated that the circularity of aggregates or liquid droplets reflects its biophysical properties, such as liquidity and aggregability, which prompted us to test the circularity of the p62-based ALIS [42].Interestingly, it turned out that YM435 significantly increased the circularity of the p62-based ALIS, meaning that YM435 increases the liquidity of the ALIS (Fig. 4F).Moreover, we found that 1,6-Hexanediol (1,6-HD), an organic compound that dissolves liquid droplet, can disaggregate the p62-based ALIS in much shorter time periods (5-10 min), when YM435 was co-treated (Fig. 4G).Considering that YM435 prevented the accumulation of p62 and K48-linked polyubiquitinated proteins in the Triton-X-insoluble fraction, YM435 appears to decrease the aggregability of the p62-based ALIS by increasing its solubility (Fig. 4G).

Aggregability of the p62-based ALIS determines the sensitivity to parthanatos
We next examined whether the solubilizing effect of YM435 on the p62-based ALIS is brought about by the structural features of YM435 or signaling mechanisms mediated by D1 dopamine receptor.Interestingly, in vitro disaggregation assay revealed that YM435 can dissolve the ubiquitinated proteins on the p62-based ALIS purified from CTX-treated HT1080 cells in vitro, suggesting that YM435 can directly solubilize the ubiquitinated proteins on the p62-baced ALIS independently of D1 dopamine receptor signaling (Fig. 5A).Therefore, we paid attention to the molecular structure of YM435.Interestingly, we found that dopamine and L-DOPA, a dopamine precursor, but not phenylalanine also could disaggregate the p62-based ALIS (Fig. 5B).As shown in Fig. 5C, two hydroxy groups on the benzene ring are common structures among YM435, dopamine and L-DOPA, a dopamine precursor.On the other hand, phenylalanine that has a structure in which two hydroxyl groups are removed from L-DOPA failed to dissolve the ALIS (Fig. 5B).This observation indicates that two hydroxy groups on the benzene ring of the dopaminergic agonists are involved in their solubilizing effect.Finally, we tested whether the solubilizing effect of the dopaminergic agonists contribute to suppress CTXinduced parthanatos mediated by the p62-based ALIS.As shown in Fig. 5D, only YM435 successfully dissolved the ALIS when lower concentrations of reagents (20 μM) were treated.Consistent with this observation, YM435 sufficiently inhibited parthanatos at the lower range (10-20 μM) (Fig. 5E).In addition, 50 μM of dopamine and L-DOPA but not phenylalanine clearly inhibited parthanatos (Fig. 5E).Thus, these observations suggest that aggregability of the p62-based ALIS determines the sensitivity to parthanatos mediated by the p62-based ALIS (Fig. 6).

DISCUSSION
In this study, we demonstrated that YM435 suppresses parthanatos through the two distinct functions: the antioxidant and disaggregating agent.Furthermore, we also demonstrated that the catecholamines such as dopamine and L-DOPA can disaggregate the p62-based ALIS.Considering that phenylalanine that has a structure in which two hydroxyl groups are removed from L-DOPA failed to disaggregate the p62-based ALIS, it is likely that two hydroxy groups on the benzene ring of YM435 are important for the disaggregation of the p62-based ALIS.Of note, the disaggregating effect of YM435 was observed at lower concentrations than the antioxidant effect of YM435, indicating that YM435 may be useful as a disaggregating agent rather than an antioxidant (Figs.1D and 3C).As previous studies have shown, these catecholamines can disaggregate the amyloid aggregates composed of amyloid β or α-synuclein [24,43].In addition, polyubiquitin and polyubiquitinated proteins have been reported to adopt amyloid-like structures [44].Therefore, YM435 may disaggregate the p62-based ALIS by acting on the amyloid-like structures of ubiquitin proteins.If so, since the abilities of YM435 to disaggregate the p62-based ALIS and to protect cells from parthanatos were more potent than dopamine and L-DOPA as shown in 5D and 5E, YM435 may be more effective in disaggregating amyloid-like structures.On the other hand, we found that YM435 suppresses cell death induced by amyloid β-peptide (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) in brain tissue (Fig. 2H).It has been reported that amyloid β-peptide (25-35) not only induces ROS generation but also forms aggregates [45].Therefore, further analysis is required to determine whether YM435's antioxidant ability of or disaggregating effect of YM435 is important for suppressing amyloid β-peptide (25-35)-induced cell death.In any case, the cytoprotective activity of YM435 may be applicable to neuroprotective therapies, although YM435 does not appear to cross the BBB as shown in Fig. 2D.Thus, further studies on drug delivery system will determine whether YM435 is appropriate as a therapeutic agent.
On the other hand, this study regarding YM435 shows that sensitivity to parthanatos is determined by aggregability of the p62-based ALIS.p62 is known to form liquid droplets with ubiquitinated proteins, which serves as the origin of selective autophagy, whereas undegraded liquid droplets tend to have solid-like properties with its maturation [46,47].Therefore, accumulation of the liquid droplets with solid-like properties (higher aggregability) seems to mean that autophagic degradation is disturbed, or droplet formation is accelerated beyond the control of autophagy.Indeed, in the presence of lysosomal stresses that disturb autophagy, p62 forms highly hydrophobic liquid droplets [48].Since these cellular conditions usually arise in pathologic and stress condition, the aggregability of the p62 liquid droplet may serve as a barometer of cellular clearance.Therefore, it seems to be reasonable that the induction of parthanatos is determined by the aggregability of the p62-based ALIS.Taken together, our Fig. 1 YM435 suppresses oxidative stress-induced parthanatos by acting as an antioxidant.A WT and PARP-1 KO HT1080 cells were treated with H 2 O 2 (1.0 mM) for the indicated periods.Cell lysates were subjected to immunoblotting with the indicated antibodies.B WT and PARP-1 KO HT1080 cells were treated with the indicated concentrations of H 2 O 2 for 5 h.Cell viability was measured by PMS/MTS assay.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; ***p < 0.001, **p < 0.01, *p < 0.05 (vs.WT).C HT1080 cells were pretreated with Rucaparib (1 μM), Pamiparib (0.1 μM), Niraparib (1 μM), Veliparib (1 μM), or E7449 (1 μM) for 1 h and then treated with H 2 O 2 (0.8 mM) for 5 h.Cell viability was measured by PMS/MTS assay.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; *** p < 0.001 (vs.DMSO).D HT1080 cells were pretreated with the indicated concentrations of YM435, Rucaparib (1 μM), or DPQ (30 μM) for 1 h and then treated with H 2 O 2 (0.8 mM) for 5 h.Cell viability was measured by PMS/MTS assay.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; ***p < 0.001 (vs.DMSO).E HT1080 cells were pretreated with YM435 (50 μM) for 1 h and then treated with H 2 O 2 (1.0 mM) for the indicated periods.Cell lysates were subjected to immunoblotting with the indicated antibodies.F HT1080 cells were pretreated with YM435 (50 μM), Rucaparib (1 μM) for 1 h and then treated with H 2 O 2 (0.8 mM) for the indicated periods.The nuclear and cytoplasm extracts were subjected to immunoblotting with the indicated antibodies.G HT1080 cells were pretreated with YM435 (50 μM), Rucaparib (1 μM) for 1 h, and then treated with H 2 O 2 (1.0 mM) for the indicated periods.The chromatin, nuclear, and cytoplasm extracts were subjected to immunoblotting with the indicated antibodies.(H) HT1080 cells were pretreated with YM435 (50 μM), NAC (2 mM) for 1 h, and then treated with H 2 O 2 (1.0 mM) for 10 min (Scale bar, 100 μm).Comet scores were calculated according to comet tail length (n = 25).I HT1080 cells were pretreated with YM435 (50 μM), DCFH-DA (10 μM) for 1 h and then treated with H 2 O 2 (1.0 mM) for the indicated periods.Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; * * * p < 0.001 (vs.DMSO).All data are representative of at least three independent experiments.investigation of the cellular response of YM435 brought about a better understanding of the biological significance of parthanatos that the p62-based ALIS with higher aggregability caused by hypoactivity of cellular clearance trigger cell death.Although it is still unknown how the p62-based ALIS with higher aggregability initiate parthanatos, our results uncovered novel aspect related to cell life and death, which may new insight into the pathogenesis of the stress-induced tissue damage and diseases.

Generation of knockout cell lines
PARP-1 and p62 knockout (KO) cells were established by using the CRISPR/ Cas9 system, and characterized as described previously [17].Guide RNAs (gRNAs) were designed to target a region in the exon 1 of PARP-1 gene (5'-GAGTCGAGTACGCCAAGAGC-3'), and that in the exon 3 of p62 gene (5'-AGACTACGACTTGTGTAGCG-3') using CRISPRdirect.Then, gRNA-encoding oligonucleotide was cloned into lenti-CRISPRv2 plasmid (addgene, Watertown, MA, USA), and the plasmid was transfected with HEK293A cells with an envelope plasmid pVSV-G and a packaging plasmid psPAX2.The supernatants were used to infect HT1080 cells, and then infected cells were cloned by limiting dilution to obtain 100% efficiency in the presence of puromycin.To check the mutations of PARP-1 and p62 in cloned cells, genomic sequence, including the target region was analyzed by PCR using extracted DNA from each clone as a template, and the following primers were used.For determination of PARP-1 mutation; 5'-GCATCAGCAATC-TATCAG-3' and 5'-CTTCCCGGACACAGTTAA-3'.For determination of p62 mutation; 5′-GAGGACTTTAGGGGGTCCCA-3′ and 5′-AGGAATTAGCA-GAGCGGCAG-3′.Cloned cell with mutations near the target sequence were established as PARP-1 or p62 KO cells (#1 or #2).

Nuclear/chromatin extraction
Nuclear/chromatin extraction was performed as described previously with minor modifications [52].Briefly, the cells were washed with phosphatebuffered saline (PBS) and pelleted at 1500x g in an eppendorf tube at 4 °C for 3 min.Pellets were resuspended by gentle pipetting in ice-cold E1 buffer [50 mM Hepes-KOH (pH 7.5), 100 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol, 0.5% NP-40, 0.25% triton X-100, 1 mM DTT, and 1% protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan)], and then centrifuged at 1,100 x g at 4°C for 5 min.The supernatant was collected in a fresh tube (cytoplasm fraction; Cy) and the pellet was resuspended by gentle pipetting in the same volume of E1 buffer and centrifuged as before, and the supernatant was discarded twice.The pellets were resuspended by gentle pipetting in ice-cold E2 buffer [10 mM Tris-HCl (pH8.0),300 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), and 1% protease inhibitor cocktails] and centrifuged at 1100x g at 4°C for 5 min.The supernatant was collected in a fresh tube (Nucleus fraction; N) and the pellet was resuspended by gentle pipetting in the same volume of E2 buffer and centrifuged as before, and the supernatant was discarded twice.The pellets were lysed in RIPA buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1% deoxycholate, and 0.1% SDS], supplemented 1% SDS and 0.02% benzonase, and collected in a fresh tube (Chromatin fraction; Ch).All Nucleus fractions; N all is a lysed sample of pellet after sampling in E1 buffer with RIPA buffer supplemented 1% SDS and 0.02% benzonase.

in vitro disaggregation assay
The insoluble fraction of the cell extract was dissolved in the 1% Triton X-100 buffer containing each compound, shaken for 30 min, and centrifuged at 15,000 rpm at 4 °C for 15 min.The supernatant was collected in a fresh tube (insoluble → soluble fraction).The pellets were lysed in RIPA buffer supplemented 1% SDS and 0.02% benzonase and collected in a fresh tube (insoluble fraction).

Colorimetric cell viability assay
Cell viability assay using Cell Titer 96 Cell Proliferation Assay (Promega, Madison, Wisconsin, USA) was performed as described previously [54].Cells were seeded on 96-well plates.After indicated stimulation or treatment, cell viability was determined according to the manufacturer's protocol.The absorbance was read at 492 nm using a microplate reader.Data are normalized to control (100%) without stimulus, unless noted otherwise.

Immunofluorescence staining
HT1080 cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 3% BSA-PBS, and incubated with primary antibodies (anti-p62, anti-ubiquitin) overnight at 4°C, followed by incubation with secondary antibodies (p62: goat anti-rabbit Alexa Fluor488, ubiquitin: goat anti-mouse Alexa Fluor 555, Invitrogen) for 1 h at room temperature.The immunostained samples were enclosed with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Nacalai Tesque), and observed using a BZ-X800L fluorescence microscope.For every sample, three images were acquired randomly, and the number, size, and circularity of ubiquitin puncta were measured by ImageJ [57].

Alkaline Comet assay
The Comet assay was performed according to the protocol of Comet SCGE assay kit (Enzo Life Sciences, Inc., NewYork, USA).Briefly, HT1080 cells are mixed with agarose in which the cells have been thawed, applied to slides, and allowed to stand at 4°C for 10 minutes.After the agarose has set, the slides were immersed in lysis solution at 4°C for 30 minutes.After that, the slides are immersed in Alkaline solution -NaOH (EDTA 1 mM, pH>13) for 30 minutes at room temperature, then electrophoresed at 25 V for 5 minutes.CyGREEN was added to the slides and stained for 30 minutes, followed by observation under a fluorescence microscope.Comet scores were calculated according to comet tail length (n = 25) [37].

Animal experiment
All mouse pups used in the study were C57BL/6.The parent mice were purchased from CLEA Japan (Shizuoka, Japan).Mouse brain slice cultures were prepared according to the previously reported preparation method [58].Briefly, 1 h before dissecting the mice, 600 µL of slice culture medium (50% Opti-MEM, 25% Hanks' Balanced Salt Solution [HBSS], 25% heatinactivated horse serum, 2 mM L-glutamine, 6.5 mg/mL glucose, and 1% penicillin-streptomycin solution) were added to each well of a 12-well plate, and incubated in 5% CO 2 at 37°C.The cerebral cortices of the mouse pups (P7) were dissected and immediately placed in ice-cold Dulbecco's Modified Eagle Medium (DMEM), for 5 min.The cerebral cortices were cut into 350 µm-thick sagittal slices using a McIlwain Tissue Chopper (Mickle Laboratory Engineering, Cambridge, UK) and incubated on ice for 30 min in DMEM.A slice was placed on the culture plate insert of the 12-well plate and maintained in 5% CO 2 at 37°C.The incubation media were replaced after 24 h and used for after 3 days.Each experiment was started 10 days after slicing.In order to test whether YM435 crosses the BBB, 1 mM YM435 or veliparib dissolved in PBS was infused into the jugular vein of C57BL mice (n = 4) for 1 hour (200 μL / hr) under general anesthesia, and then plasma and brain were collected.The concentration of YM435 or veliparib was measured by using LC / MS / MS as previously described [59].

PI staining
After each stimulation, mouse brain slices were stained with PI (5 µg/mL) in the medium for 1 h.Slices were fixed with 3.7% formaldehyde, and then enclosed with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Nacalai Tesque), and observed using a BZ-X800L fluorescence microscope.For every slice, five images were acquired randomly, and the number of PI puncta were counted for each image.

Statistical analysis
The value was expressed as the mean ± standard deviation (S.D.) using Prism software (GraphPad).All experiments were repeated at least three independent times.Two groups were compared using student's t-test.Multiple-group comparisons were conducted using the one-way ANOVA analysis of variance followed by the Tukey-Kramer test using Prism software (GraphPad).Data were considered significant when *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 6 Schematic model to explain our study.The excessive accumulation of ROS initiates oxidative stress, leading to the formation of the p62-based ALIS.In the case of the p62-based ALIS with higher liquidity, the p62-based ALIS is basically nontoxic, and may be degraded by the autophagic processes.On the other hand, the p62-based ALIS with lower liquidity and higher aggregability display cytotoxicity through the induction of parthanatos.The D1 dopamine receptor agonist YM435 reduces aggregability of the p62-based ALIS, and change the cytotoxic ALIS to the nontoxic ALIS.Therefore, YM435 serves as an inhibitor of parthanatos induced by the p62-based ALIS.

Fig. 3
Fig. 3 YM435 suppresses parthanatos independently of its antioxidant activity.A WT and PARP-1 KO HT1080 cells were treated with the indicated concentrations of CTX for 36 h.Cell viability was measured by PMS/MTS assay.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; ***p < 0.001 (vs.WT).B HT1080 cells were treated with YM435 (20 μM), DPQ (30 μM), Rucaparib (1 μM) and CTX (0.6 mg/mL) for 36 h.Cell viability was measured by PMS/MTS assay.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; ***p < 0.001, (vs.DMSO).C HT1080 cells were treated with the indicated concentrations YM435 and CTX (0.6 mg/mL) for 36 h.Cell viability was measured by PMS/MTS assay.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; **p < 0.01, ***p < 0.001 (vs.DMSO).D HT1080 cells were treated with the indicated concentrations YM435 and CTX (0.6 mg/mL) for 28 h, and then incubated with DCFH-DA (10 μM) for 30 min.Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; N.S.: not significant [vs.YM435 (0 μM)].E HT1080 cells were treated with the indicated concentrations YM435, NAC (2 mM) and CTX (0.8 mg/mL) for 24 h.The nuclear and cytoplasm extracts were subjected to immunoblotting with the indicated antibodies.F HT1080 cells were treated with the indicated concentrations YM435, NAC (2 mM) and CTX (0.8 mg/mL) for 24 h, and then the mRNA levels were measured by quantitative real-time PCR.Data shown are the mean ± SD (n = 3).Statistical significance was tested by one-way ANOVA, followed by Tukey-Kramer test; ***p < 0.001, *p < 0.05, N.S.: not significant [vs.YM435 (0 μM)].G HT1080 cells were treated with YM435 (20 μM) and CTX (1.0 mg/mL) for the indicated periods.Cell lysates were subjected to immunoblotting with the indicated antibodies.All data are representative of at least three independent experiments.